Cd3 pacific orange

PBMCs at 1 × 10⁶ cells/100 μL were stimulated with peptide pools (F + G + H = NS3/4, I + L + M = NS5A/B) or phorbol 12‐myristate 13‐acetate/ionomycin (50 and 500 ng/mL, respectively) or unstimulated (DMSO, 5 ng/mL). Brefeldin‐A was added (10 μg/mL) 2‐4 hours later, and cells were incubated overnight (37°C), stained with fixable‐near infrared live/dead dye (Life Technologies), fixed, and permeabilized (Foxp3 Fix/Perm kit; BD Biosciences) with CD3‐Pacific Orange (1:50), CD4‐Qdot605 (1:200), CD8‐peridinin chlorophyll protein Cy5.5 (1:200), IFN‐γ‐AlexaFluor 700 (1:50), interleukin‐2 (IL‐2)‐allophycocyanin (1:25), tumor necrosis factor‐α (TNF‐α)‐phycoerythrin‐Cy7 (1:25), and IL‐17‐phycoerythrin (1:100; Becton Dickson Sciences and Invitrogen) in perm buffer (30 minutes). Flow cytometry was performed on a BD LSRII machine and analyzed using FlowJo v.9.5.2 (Treestar).

Cell surface expression was assessed on freshly isolated TILs or PBMCs by staining with the following monoclonal antibodies: CD3-Pacific Orange (Clone UCHT1, Cat. 561416, BD Biosciences, San Jose, CA, USA), CD4-APC-Cy7 (Clone OKT4, Cat. 317418, Biolegend, San Diego, CA), CD8-FITC (Clone RPA-T8, Cat. 555366, BD Biosciences, San Jose, CA, USA), CD25-Phycoerythrin (Clone M-A251, Cat. 555432, BD Biosciences, San Jose, CA, USA) and PD-1-Pacific Blue (Clone EH12.2H7, Cat. 329916, Biolegend, San Diego, CA). For detection of intracellular expression of FOXP3, cells were permeabilized and fixed with the ‘FOXP3 Staining Buffer Set’ (BD Biosciences, San Jose, CA, USA) following the manufacturer’s instructions, and then they were incubated with the anti-FOXP3-PE-Cy7 monoclonal antibody (Clone PCH101, Ref: 25-4776-42, BD Biosciences, San Jose, CA, USA). Cell analysis was performed with a FACSCanto flow cytometer (BD Biosciences, San Jose, CA, USA).